ALBERT

Description: Insulator elements are found at the boundary between euchromatin and heterochromatin, and are responsible for maintaining the correct chromatin configuration for a locus. The best characterized vertebrate insulator element, 5′ Hypersensitive Site (HS) 4 from the chicken β-globin locus (ch5′HS4), has two separable activities: enhancer blocking, which requires binding of the transcription factor CTCF, and barrier, which prevents transgene silencing. We have previously reported that transgenic mice carrying a wild-type erythrocyte ankyrin promoter (ANK-1E)/γ-globin gene showed uniform (γ-globin in 100% of red cells), position-independent (32/32 lines express), copy number-dependent (p=0.0005) expression of γ-globin mRNA and protein. Mutations in the ANK-1E promoter at positions −108 and −153 cause ankyrin-deficient Hereditary Spherocytosis. Transgenic mice with the −108/−153 ANK-1E/γ-globin transgene showed variegated (γ-globin in 0–80% of red cells), position-dependent (8/14 lines express), copy number-independent (p=0.27) expression of γ-globin. Flanking the −108/−153 ANK-1E/γ-globin transgene with the ch5′HS4 insulator restored uniform, position-independent (9/9 lines), copy number-dependent expression (p=0.003) at levels identical to the wild-type ANK-1E promoter. We hypothesized that we could test sequences for barrier activity by assaying their ability to restore normal expression to the −108/−153 ANK-1E/γ-globin gene in transgenic mice. In mammalian β-globin loci, human 5′HS5 and mouse 3′HS1 have been proposed to be insulator elements, similar to chicken 5′HS4, based on their ability to block enhancer element function. To test barrier function, we generated transgenic mice containing the −108/−153 ANK-1E/γ-globin transgene flanked by human 5′HS5, mouse wild-type 3′HS1, or mouse 3′HS1 with mutations that disrupt the binding of CTCF (×CTCF). A total of 5 lines of transgenic mice were generated containing the 5′HS5/−108/−153 ANK-1E/γ-globin transgene. γ-globin mRNA and protein were undetectable in 3/5 lines, indicating that expression was position-dependent, and in the two positive lines, mRNA levels did not correlate with copy number. A total of 9 lines of 3′HS1-flanked transgenic mice were generated, 3 of which did not express γ-globin mRNA and protein, demonstrating position-dependent expression. Among the 6 expressing lines, two lines showed variegated expression and the correlation between γ-globin mRNA level and copy number was significant (p=0.0117). In contrast, 3′HS1×CTCF transgenic mice expressed γ-globin in a uniform, position-independent (7/7 lines express), copy number-dependent (p=0.0005) manner. The levels of γ-globin mRNA in both the 3′HS1 and 3′HS1×CTCF transgenic mice were 2-fold greater than the levels measured in transgenic mice with the wild-type ANK-1E promoter (p=0.019; 0.0003 respectively), suggesting that 3′HS1 may contain an enhancer element. Our results indicate that while human 5′HS5 and mouse 3′HS1 block the effects of enhancer elements, neither are barrier elements as defined by the ability to prevent gene silencing. We hypothesize that the mutation of the CTCF binding sites allows the ANK-1E promoter to take greater advantage of the 3′HS1 enhancer, leading to a more uniform, position-independent, and copy number-dependent pattern of expression, as has been described for other enhancer elements in the β-globin Locus Control Region.

Description: Hmgb3 is a member of a family of chromatin-binding proteins that can alter DNA structure to facilitate transcription factor binding. We identified the Hmgb3 cDNA in a subtractive hybridization screen for transcripts that are preferentially expressed in hematopoietic stem cells. We inserted an internal ribosomal entry site–green fluorescence protein cassette into the 3′ untranslated region of the X-linked Hmgb3 locus to identify Hmgb3-expressing cells. In adult mice, Hmgb3 mRNA is detected in bone marrow cells, primitive Lin–, c-kit+, Sca-1+, IL-7Rα– cells, and Ter119+ erythroid cells. We observed that long-term repopulating ability is entirely contained in the subpopulation of Lin–, c-kitHI cells that express Hmgb3. Most common lymphoid and myeloid progenitors express Hmgb3. Introduction of a retrovirus containing the Hmgb3 cDNA into mouse bone marrow stem cells demonstrated that enforced expression of Hmgb3 inhibited B-cell and myeloid differentiation. We conclude that down-regulation of Hmgb3 protein levels is an important step for myeloid and B-cell differentiation.

Description: Erythrocyte ankyrin forms the bridge between the spectrin/actin cytoskeleton and the red cell membrane protein band 3. Mutations resulting in defective or deficient ankyrin are the most common cause of Hereditary Spherocytosis. The erythroid ankyrin promoter (ankR) lies between two other ankyrin promoters, one that is active in neuronal cells and the other in many cell types. Active genes are generally found in DNase I sensitive regions of chromatin while regulatory sequences (promoters, enhancers, boundary elements) are associated with DNase I Hypersensitive Sites (HS). We hypothesized that the chromatin surrounding ankR determines its activity. In human K562 cells and mouse fetal liver cells a DNase I HS (5′HS) maps between −300 and −100 of ankR. 5′HS marks the end of a DNase I resistant chromatin extends at least 10 kb upstream, indicating that this chromatin is inactive in erythroid cells. We have previously shown that a 300 bp fragment containing ankR was sufficient to direct erythroid specific, copy number dependent, position independent and uniform expression of a linked γ-globin gene in transgenic mice. Identical results were obtained with mice containing −2.7 kb and −656 bp ankyrin promoters, as expected, and 5′ HS was present in fetal liver chromatin of all 8 lines examined. Downstream of 5′HS is a 5.7 kb DNase I sensitive region that includes a 500 bp enhancer-like element near the 3′ end. At the 3′ end of the DNase I sensitive domain are 2 HS (3′HS) that mark the beginning of a DNase I resistant region that extends at least 3 kb downstream. AnkR is inactive in non-erythroid HeLa, Jurkat and Sh-SY5Y (neuronal) cells. Although both 5′HS and 3′HS are present in these cells, the intervening 5.7 kb region is DNase I resistant indicating that erythroid specific factors are required for the activation of ankR. Boundary elements (insulators) are found between DNase I resistant and DNase I sensitive chromatin and have the ability to prevent the transgene silencing at ectopic sites in the genome. To test whether the 5′HS and 3′HS function as boundary elements, a β-globin promoter/EGFP transgene was flanked by 200 bp fragments containing either 5′HS or 3′HS. While 0/8 K562 cell lines with the transgene alone expressed GFP, GFP expression was observed in 12/12 5′HS and 11/12 3′HS K562 cell lines (χ2=12.0, p

Description: Alpha-Hemoglobin Stabilizing Protein (AHSP) is an erythroid-specific protein that binds to α-globin, preventing precipitation of α-hemoglobin tetramers. Our interest has focused on how the AHSP gene is specifically expressed in erythroid cells, and we have investigated the roles of cis acting DNA sequences, the transcription factor EKLF and chromatin structure on AHSP gene expression. We have previously shown that the AHSP gene has a single mRNA initiation site followed by a non-coding exon. Putative promoter sequences from −904, −479 or −170 were active in luciferase reporter assays only when the constructs contained the downstream 269 bp containing exon 1 and intron 1. The −904/+269, −479/+269 and −170/+269 constructs gave 53.3+2.0 to 122.1+8.8 -fold increased levels of luciferase expression in K562 cells compared to plasmids without exon 1 and intron 1 (p

Description: Hmgb3 is an X-linked member of a family of sequence-independent chromatin-binding proteins that is expressed in HSC-enriched lin−, c-kitHI, Sca-1HI, IL-7Rα− (KSIL) cells and Ter119+ erythroid cells. To define Hmgb3 function, we generated hemizygous mice (Hmgb3−/Y) using 129/SvJ ES cells. Hmgb3−/Y mice contain normal numbers of KSIL cells that are capable of normal repopulation and self-renewal. However, these mice have 1.6-fold fewer common lymphoid progenitors (CLP) and 3-fold fewer common myeloid progenitors (CMP) (p 〈 0.05). We hypothesized that the role of Hmgb3 in early hematopoiesis involves c-kit regulation. We observed that the level of c-kit mRNA in Hmgb3−/Y HSCs increased 30% compared to wild-type (WT) (p = 0.05). We used 5-fluorouracil (5-FU), which has been shown to down-regulate c-kit on HSCs, to characterize the interaction between Hmgb3 and c-kit. We monitored Hmgb3 expression in KSIL and lin−, Sca-1+, c-kit− cells before and after 5-FU treatment (150 mg/kg) using phenotypically normal transgenic mice containing an IRES-GFP cassette knocked into the 3′ UTR of Hmgb3. Prior to 5-FU treatment, 27% of KSIL cells were GFP+ (these cells were absent 4 days post-injection {p.i.}). In contrast, 1.8% of lin−, c-kit−, Sca-1+ cells were GFP+ before 5-FU treatment whereas 26% of lin−, c-kit−, Sca-1+ cells were GFP+ 4 days p.i. The increased proportion of GFP+ lin-, c-kit−, Sca-1+ cells after 5-FU treatment is consistent with previous findings that repopulating activity resides within the c-kit−/LO population in 5-FU treated bone marrow and our finding that Hmgb3 serves as a marker for long-term repopulating activity. To determine the time course of c-kit regulation, we compared bone marrow from 5-FU injected Hmgb3−/Y and WT mice for analysis at 2, 4, and 6 days p.i. Two days p.i., both WT and Hmgb3−/Y mice contained similar numbers of bone marrow cells (7 x 106 cells/hind limb) and the KSIL population was absent. By four days p.i., the bone marrow cellularity of WT mice declined to 5.5 ± 0.9 x 106 cells/hind limb and KSIL cells were still absent. However, in Hmgb3−/Y mice 4 days p.i., bone marrow cellularity stabilized at 7.9 ± 0.8 x 106 cells/hind limb, an increase of 43% compared to WT (p 〈 0.01), along with the re-emergence of the KSIL population. To determine whether the Hmgb3−/Y lin−, c-kit−, Sca-1+ population contains repopulating HSCs after 4 days of 5-FU treatment similar to WT mice, we performed repopulation assays using KSIL and lin−, c-kit−, Sca-1+ cells sorted from 4 day p.i. 5-FU treated Hmgb3−/Y mice. Recipients received either 2 x 104 KSIL or 2 x 105 lin−, c-kit−, Sca-1+ cells (Ly 5.2) from 5-FU treated Hmgb3−/Y mice along with a radioprotective dose of 3 x 105 congenic (Ly 5.1) bone marrow cells. FACS analysis performed on control recipients transplanted with congenic marrow exhibited 〈 1% Ly 5.2 cells in the bone marrow 16 weeks after transplant. Pre-5-FU treatment, 88% of bone marrow cells were donor derived in recipients of Hmgb3−/Y KSIL cells. There was no detectable engraftment of Hmgb3−Y lin−, c-kit−, Sca-1+ cells. In contrast to WT mice, both KSIL and lin−, c-kit−, Sca-1+ cells from 5-FU treated Hmgb3−/Y mice were capable of long-term repopulation (62–82% donor derived cells). We conclude that Hmgb3 deficiency facilitates the reemergence of c-kitHI HSCs following 5-FU treatment. Mechanisms involving either enhanced HSC self-renewal or delayed differentiation into CLPs and CMPs are both consistent with our results.

Description: Hmgb3 is an X-linked member of a family of chromatin-binding proteins that is expressed in primitive hematopoietic cells capable of long-term hematopoietic repopulation. To examine the role of Hmgb3 in adult hematopoiesis, we generated Hmgb3-deficient (Hmgb3–/Y) mice, which are viable but erythrocythemic. Hmgb3–/Y mice contain normal numbers of hematopoietic stem cells (HSCs), which generate fewer than normal numbers of common lymphoid progenitors (CLPs) and common myeloid progenitors (CMPs) and greater than normal numbers of more mature progenitors. Although fewer Hmgb3–/Y primitive progenitor cells are in the G2/M cell cycle phase, bromodeoxyuridine (BrdU) incorporation demonstrated enhanced proliferation compared with their wild-type counterparts. Hmgb3–/Y HSCs have increased levels of Gata-2 and c-myb mRNA. We propose that Hmgb3 deficiency leads to a failure of HSCs to expand into normal numbers of CLPs and CMPs. This defect is compensated for by the ability of Hmgb3–/Y progenitors to expand rapidly and differentiate into normal numbers of hematopoietic cells.